Many DNA isolation techniques
widely employed by plant molecular biologists use a CTAB (hexadecyltrimethylammonium
bromide) extraction buffer coupled with reusable tissue homogenization
systems such as a mortar and pestle (3,4,10). These procedures, though simple, typically use
large amounts of buffer (10 ml), utilize nondisposable homogenizers and
require ethanol washes. The risk of cross contamination associated with
reusing homogenizers and vessels is unacceptable if the DNA isolated will
be amplified in PCR or RAPD (random amplified polymorphic DNA, 11)
experiments. Recent DNA extractions methods developed to avoid potential
contamination disrupt cells by biochemical means (1),
leaf squashes (7) or sodium dodecyl sulfate mini
preps (5). However, the biochemical lysis method
and the leaf squash method are complicated and/or do not yield sufficient
DNA for many replicate reactions. The SDS procedure is similar to the
protocol described here, but the CTAB buffer should be more amenable to
plant material containing polysaccharides (4, 6).

The procedure presented is
a modification of the Doyle and Doyle CTAB method (4)
scaled to fit in microcentrifuge tubes with reagents (6)
added to increase separation of polysaccharides from the DNA. Milligram
amounts of leaf tissue is ground using a cordless drill driven pipette
tip (as devised by authors) in a microcentrifuge tube with hot CTAB buffer.
A single chloroform isoamyl alcohol (24:1) extraction is followed by a
single isopropanol precipitation. This simplified, quick, and inexpensive
CTAB procedure yielded sufficient template for >100 reactions using
only disposable homogenizers and vessels, and requiring only one transfer
of the DNA solution thereby reducing potential DNA cross contamination.
The RAPD fingerprinting technique is thought to be sensitive to the quality
of the DNA template (11). To show the success of
the presented CTAB mini prep method, DNA samples were isolated from 5
plant and 1 fungus species by both the Doyle and Doyle method (4)
followed by purification through a CsCl ethidium bromide gradient and
the described miniprep procedure. The DNA samples isolated by the two
methods were used in RAPD reactions and the fingerprints compared. The
DNA isolated by the described CTAB miniprep method compared favorablely
to the control CsCl cleaned DNA for use in RAPD reactions.

MATERIALS AND METHODS

DNA was extracted from 5 plant
species (American chestnut, Castanea dentata; American cranberry,
Vaccinium macrocarpon; geranium, Pelargonium x hortorum;
Peters Mountain mallow, Iliamna corei; and peanut, Arachis hypogaea)
and 1 fungus (Russula sp.) by two methods. A standard CTAB genomic
DNA isolation method (4) followed by ultracentrifugation
through a cesium chloride/ethidium bromide gradient (8)
was used as a control isolation method against which the following method
was judged. For the control method 0.33 grams of plant or fungal tissue
was processed in 10 ml CTAB buffer and the resulting DNA was resuspended
in 100 l of TE (10 mM Tris HCl, 1 mM EDTA, pH 7.4).

For the CTAB mini prep method,
an Eppendorf 1000 l plastic pipette tip that had been pushed onto a deburring
tool mounted on a cordless drill served as the homogenizing pestle. The
end of the pipette tip was crimped upward when pressed on the tool (the
tip was pressed against the bottom of the pipette tip box), thereby creating
a "blade" for homogenization (Fig. 1). Fresh leaf (or stipe)
tissue (~.025 g) was placed in 600 l 70 C extraction buffer (2% w/v CTAB,
1.42 M NaCl, 20 mM EDTA, 100 mM Tris HCl pH 8.0, 2% w/v PVP 40 {polyvinylpyrrolidone}
(Sigma Chemical Co., St. Louis, MO), 5 mM Ascorbic acid, 4.0 mM DIECA
{diethyldithiocarbamic acid} (Sigma Chemical Co., St. Louis, MO), 4)
in a microcentrifuge tube. PVP ascorbic acid, and DIECA additions were
not used in the control. Just prior to homogenization, 3 l of 2 mercaptoethanol
was added to the tube. Immediately following homogenization (600 rpm for
20 s), the homogenate was extracted with 500 chloroform isoamyl alcohol
(24:1 v/v). This mixture was shaken for 5 min at 500 rpm (IKA Vibrax VXR,
Cincinnati, OH) and centrifuged (1000 x g at 22 C) in microcentrifuge
for 5 min to separate phases. The upper, aqueous, DNA containing phase
was transferred to a fresh microcentrifuge tube, precipitated with 0.7
volume isopropanol for 5 min at 22 C and centrifuged (14,000 x g) for
20 min. The pellet was air dried and resuspended in 100 l of TE.

Yields from the control isolation
procedure (4) were 50 to 120 g of DNA. DNA yields
from the CTAB mini preparation ranged from 20 Russula and Chestnut) to
34 g (Mallow). This represents enough DNA to do 100-400 typical RAPD reactions.
DNA yields per gram of plant tissue from the control isolation procedure
were 150 to 360 g/g and the CTAB mini prep yields were 80 to 140 g/g plant
tissue. While the DNA yield from the control method was 66% higher, the
PCR fragment patterns were not different between isolation techniques
for each tissue sample (Fig. 2). RAPD analysis employing other 10 base
primers gave equivalent results for the two methods of DNA preparation
(data not shown).

When doing population studies
using RAPD, often the time consuming step is isolating DNA from numerous
samples. Three days were required to process 6 samples by the control
CTAB procedure (4) including a CsCl gradient. Twenty to thirty samples
can be processed per day using the Doyle and Doyle procedure without the
CsCl gradients. In contrast, greater than a hundred tissue samples can
be processed in a day using the CTAB mini prep procedure. For researchers
using amplification techniques on hundreds of plant DNA samples, large
yields are likely to be less important than speed and cost of sample preparation.
Additionally, polysaccharides, which are abundant in peanut and mallow
leaves are known to be bound by PVP (4), thus eliminating
the need for additional removal methods for these compounds (2,
6). Since the method uses disposable homogenizers,
and is done in microcentrifuge tubes there is diminished possibility for
cross contamination between samples. Care should be taken to homogenize
samples individually in a chemical hood with adequate air flow to protect
both the researcher and the DNA preparation. However, this procedure,
with minor modifications in equipment, may useful for DNA extractions
in the field. This procedure can be directly applied to many different
plants species from polysaccharide rich mallow and peanut to leathery
leaved cranberries.

ACKNOWLEDGEMENTS

This work was performed in
the laboratories of Joseph O. Falkinham, III and Erik T. Nilsen. Financial
support was provided by Public Health Service Grant AI 30373 from National
Institute of Allergy and Infectious Disease and a grant from the Heiser
Foundation, Inc. Susan Stewart's drawing of Figure 1 is greatly appreciated.

REFERENCES

1. Deragon,
J.M. and B.S. Landry. 1992. RAPD and other PCR based analyses of plant
genomes using DNA extracted from small leaf discs. PCR Methods and
Applications 1:175-180.

Figure 1.
The homogenizer: A deburring tool (A) is pushed onto a racked, plastic
1000 ml Eppendorf pipette tip (any brand should should suffice). This
forces the end of the pipette tip to be bent at a ~45 angle. The deburring
tool's conical head had the following dimensions: 9 mm maximum outside
diameter and 20 mm in length.

Figure 2.
Comparison of RAPD patterns of DNA prepared by the CTAB, CsCl gradient
method and the CTAB miniprep method described here. Lane 1 contains the
100 bp marker (Gibco BRL, Gaithersburg, MD). In each of the following
pairs of lanes the CsCl gradient purified DNA is presented first followed
by the CTAB mini prep DNA. The samples presented are American cranberry
(lanes 2 and 3), Peters Mountain mallow (lanes 4 and 5), American chestnut
(lanes 6 and 7), geranium (lanes 8 and 9), peanut (lanes 10 and 11) and
Russula (lanes 12 and 13) amplified with primer OPA 04 5'AATCGGGCTG (Operon
Tech. Inc., Alameda, CA).